Effects of joint screening for prostate, lung, colorectal, and ovarian cancer – results from a controlled trial

Background Although screening is widely used to reduce cancer burden, untargeted cancers are frequently missed after single cancer screening. Joint cancer screening is presumed as a more effective strategy to reduce overall cancer burden. Methods Gender-specific screening effects on PLCO cancer incidence, PLCO cancer mortality, all-neoplasms mortality and all-cause mortality were evaluated, and meta-analyses based on gender-specific screening effects were conducted to achieve the pooled effects. The cut-off value of time-dependent receiver-operating-characteristic curve of 10-year combined PLCO cancer risk was used to reclassify participants into low- and high-risk subgroups. Further analyses were conducted to investigate screening effects stratified by risk groups and screening compliance. Results After a median follow-up of 10.48 years for incidence and 16.85 years for mortality, a total of 5,506 PLCO cancer cases, 1,845 PLCO cancer deaths, 3,970 all-neoplasms deaths, and 14,221 all-cause deaths were documented in the screening arm, while 6,261, 2,417, 5,091, and 18,516 outcome-specific events in the control arm. Joint cancer screening did not significantly reduce PLCO cancer incidence, but significantly reduced male-specific PLCO cancer mortality (hazard ratio and 95% confidence intervals [HR(95%CIs)]: 0.88(0.82, 0.95)) and pooled mortality [0.89(0.84, 0.95)]. More importantly, joint cancer screening significantly reduced both gender-specific all-neoplasm mortality [0.91(0.86, 0.96) for males, 0.91(0.85, 0.98) for females, and 0.91(0.87, 0.95) for meta-analyses] and all-cause mortality [0.90(0.88, 0.93) for male, 0.88(0.85, 0.92) for female, and 0.89(0.87, 0.91) for meta-analyses]. Further analyses showed decreased risks of all-neoplasm mortality was observed with good compliance [0.72(0.67, 0.77) for male and 0.72(0.65, 0.80) for female] and increased risks with poor compliance [1.61(1.40, 1.85) for male and 1.30(1.13, 1.40) for female]. Conclusion Joint cancer screening could be recommended as a potentially strategy to reduce the overall cancer burden. More compliance, more benefits. However, organizing a joint cancer screening not only requires more ingenious design, but also needs more attentions to the potential harms. Trial registration NCT00002540 (Prostate), NCT01696968 (Lung), NCT01696981 (Colorectal), NCT01696994 (Ovarian).


Introduction
Cancer ranks as the second leading cause of death and the primary cause of years of life lost, years lived with disability and disability-adjusted life-years in several countries around the world (1)(2)(3).An estimated 19.3 million new cancer cases and almost 10 million cancer deaths occurred in 2020 (1).Due to the rapidly growing epidemic of several cancer-associated risk factors, including aging, tobacco use, unhealthy diet, excess body weight, physical inactivity, and air pollution, the cancer burden is expected to grow in most parts of the world, particularly in transitioning or developing countries (1,4,5).Addressing the growing global burden of cancer has become a global concern.
For more than a half of century, cancer screening has been widely used as an effective strategy to reduce the cancer burden in several countries.Until now, several high-level evidences had supported that population-based screening for cancer at different sites (including colorectum, cervix uteri, lung, breast, and prostate) can reduce cancer-specific mortality by about 18%-20% (6)(7)(8)(9)(10)(11)(12), and similar conclusions can be observed across different periods (13)(14)(15)(16)(17).Even in countries with limited resources, one-time populationbased mass screening has also been shown to be associated with a significant reduction in cancer-specific incidence and mortality (18)(19)(20).Other research efforts are also underway to reduce the burden of cancers with relatively low incidence and poor prognoses, such as ovarian cancer and liver cancer (21,22).However, in both well-resourced and resource-limited areas, previous and current cancer screening programs just focus on one site of cancer at a time (6-12, 23, 24).Untargeted cancers are often missed and later reported after a single cancer screening.To reduce the burden of cancer uncovered by the previous screening program, policymakers have to organize another independent screening program for uncovered cancer.When this demand involves several cancers at multiple sites, duplication of investment and waste of limited resources will be inevitable in the context of a single cancer screening strategy.Therefore, a new screening mode, namely joint cancer screening, has been raised as a more effective screening strategy to reduce the overall cancer burden than the current single cancer screening strategy (25).Joint cancer screening means that individuals in the screening group received multiple methods recommended by the guidelines to screen for different cancers during the same period.More importantly, risk-stratified joint cancer screening would be a more cost affordable or sustainable screening strategy for regions or countries with large populations but limited resources.However, few studies have investigated the effect of joint cancer screening, and fewer studies have investigated the effect of risk-stratified joint cancer screening.
The Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial is currently the only published randomized, controlled trial (RCT) in the world to screen prostate, lung, colorectal and ovarian cancer concurrently in the same population (26).The study had previously reported the screening effectiveness for four targeted cancers (9,(27)(28)(29).However, no study has evaluated the effectiveness of joint cancer screening based on its exclusive "all-versus-none" design, especially the effectiveness of risk-stratified joint cancer screening based on additional follow-up and outcomes.Therefore, this study aims to investigate the effectiveness of joint cancer screening on PLCO cancer incidence, PLCO cancer mortality, all-neoplasms mortality, and all-cause mortality, and try to develop a risk-stratified joint cancer screening strategy suitable for countries with increasing cancer burden and limited resources.

Method Source of population and selection of participants
The design and protocol of the PLCO cancer screening trial have been described previously (30).Briefly, the PLCO cancer screening trial is an RCT that enrolled 155,000 participants aged 55 to 74 between November 1993 and July 2001.Participants were individually randomized to the control arm or intervention arm in equal proportions.Participants assigned to the control arm received usual care, whereas participants assigned to the intervention arm received screening exams for prostate, lung, colorectal, and ovarian cancers as outlined in the study protocol.Men and women received organ-specific screening protocols, respectively.Men received a combination of a six-round annual prostate-specific antigen (PSA) exam and a four-round annual digital rectal examination (DRE) to detect prostate cancer.Women received a combination of six-round annual cancer antigen 125 (CA-125) and four-round annual transvaginal ultrasound (TVU) to detect ovarian cancer.Both men and women were given a four-round annual posteroanterior chest X-ray (XRY) to detect lung cancer and tworound flexible sigmoidoscopy (FSG) with an interval of three to five years to detect colorectal cancer.
In this study, a total of 149,161 eligible participants were initially included after excluding 4,918 participants with no baseline questionnaire returned, 185 participants with a history of PLCO cancer, and 623 participants lost to follow-up.After further excluding 10,322 participants with missing data of index predictors of PLCO cancer and 6,778 participants with a history of any other cancers, a total of 66,490 male participants and 65,533 female participants were selected for further analyses.In the screening arm, there were 1,635 male participants and 2,619 female participants who did not receive any screening, these individuals who were non-compliant with screening were compared to those who were compliant with screening in sensitivity analyses based on screening compliance.Furthermore, after excluding participants who did not received at least one-round joint cancer screening, 61,248 male participants and 57,240 female participants were finally included in the preliminary analyses.Detailed flowchart referred to Supplementary Figure 1.
Participants who had received a positive screening result were encouraged to receive a further diagnostic evaluation.Participants and their physicians decided which procedures would be performed.In the case of a positive FSG, it was standard practice not to biopsy or remove polyps or masses; instead, a referral was made to the participant's physician for diagnostic evaluation and follow-up.

Cancer diagnosis and primary outcomes
The PLCO trial confirmed diagnoses of cancer through medical record abstraction (MRA), which was triggered by (9,(26)(27)(28)(29): 1) self-report of cancer on an Annual Study Update (ASU); 2) positive screening exams mentioned above; 3) death certificate indicating cancer; 4) Death Review Process (DRP) suspected cancer based on other indicators; 5) relative informs screening center of participant's cancer.Cancer diagnosis was followed up to December 31, 2009 and mortality was followed up to December 31 2018.For every cancer that was ascertained, information on the cancer diagnosis date and ICD-O-2 code was collected and recorded on the appropriate medical record abstract form.For primary PLCO cancer ascertained, additional information on diagnostic procedures, cancer stage, grade, histopathologic type, and initial cancer treatment was further recorded.
The primary outcomes of this study included PLCO cancers incidence, PLCO cancers mortality, all-neoplasms mortality and allcause mortality.To assess these endpoints, all deaths from any cause were collected and ascertained according to the following main sources: 1) the administration of the ASU questionnaires, 2) reports from relatives, friends, or physicians, and 3) National Death Index (NDI) plus searches.After notification, PLCO Screening Centers attempted to obtain a death certificate for each death before 2018.The underlying leading cause of death was derived according to rules established by the National Center for Health Statistics.To provide a more accurate assessment of the trial endpoints, a DRP was conducted and medical records were reviewed for all deaths that might have been due to PLCO cancers.The DRP cause of death was considered authoritative and was used in statistical analyses of the primary endpoints.

Assessment of covariates
After informed consent, all participants were provided with a baseline questionnaire to collect participant-reported information on demographic and potential risk factors associated with PLCO cancers, including age, sex, race, height, weight, smoking status, medical history (including diabetes, hypertension, heart attack, stroke, bronchitis, emphysema, liver comorbidity, colon comorbidity, polyps, diverticulitis), regular use of non-steroidal anti-inflammatory drugs (NSAIDs) (including aspirin and ibuprofen) in the last 12 months, sex-specific information (such as oral contraceptives and hormone replacement therapy for female, and enlarged prostate, prostatitis, and prostate surgery for male), personal history of cancer (including PLCO cancers and any other cancers), personal screening history of PLCO cancer, and family history of any cancer among first-degree relative.Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m 2 ).

Statistical analysis
Chi-square tests were used to compare the distribution of potential confounding factors associated with screening effectiveness between the screening arm and control arm.Genderspecific cumulative PLCO cancer incidence, PLCO cancer mortality, all-neoplasms mortality, and all-cause mortality were calculated with Kaplan-Meier curves and compared with log-rank tests.Gender-specific screening effectiveness of primary endpoints was evaluated with multivariable Cox regression analyses after adjusting all available confounding factors.Due to different genderspecific factors associated with primary outcomes, meta-analyses based on gender-specific screening effectiveness rather than multivariable Cox regression analyses adjusting gender were conducted to achieve the pooled effectiveness.The I² statistic was calculated to determine the heterogeneity between gender-specific screening effectiveness (31).Subgroup analyses based on combined PLCO cancer risk assessment and sensitivity analyses by screening compliance were further conducted to explore the effectiveness of risk-stratified joint cancer screening.
To develop a gender-specific PLCO cancer risk prediction model (PLCO-CA model) for the general population without intervention, 70% of the participants in the control arm were randomly assigned to the derivation cohort (22,726 male participants and 22,798 female participants), while the remaining 30% were assigned to the validation cohort (9,778 male participants and 9,772 female participants).Since diabetes has been extensively studied concerning the increased risk of developing cancer (32), diabetes has been listed as the potentially independent predictor of PLCO cancer.History of organ-related partial resection surgery would suggest a greater potential risk of cancer than other benign organ-related diseases, so it was also used as a potentially independent predictor of PLCO cancer risk.To achieve relatively stable and easy-to-use sex-specific PLCO-CA models, chronic bronchitis, emphysema, polyps, diverticulitis or diverticulosis, colon comorbidity, prostatitis (male), enlarged prostate or benign prostatic hypertrophy (male), and benign ovarian tumor/cyst (female) were combined and defined as organ-related diseases (0, 1, ≥2).Stroke, heart attack, hypertension, and liver comorbidity were combined and defined as non-organ-related diseases (0, 1, ≥2).All available PLCO cancer-associated sex-specific predictors were listed in Supplementary Tables 1, 2, and they were initially selected to develop sex-specific PLCO-CA models.
In the derivation cohort, univariate Cox regression was first used to test the crude association of PLCO cancer with each potential predictor.The potential predictors with P < 0.25 were preliminarily selected into multivariable Cox regression.P value < 0.05 for entry and P value > 0.10 for removal were used in the both forward and backward variable selection procedures within the multivariable Cox regression to select independent predictors of PLCO cancers.The hazard ratios (HR) and its 95% confidential interval (CI) were calculated to measure the association between predictor and PLCO cancer risk.The discriminations of the genderspecific PLCO-CA models were evaluated by the receiveroperating-characteristic curves (ROCs) and the areas under the curve (AUCs).The calibrations of PLCO-CA models were assessed by calibration curves and measured as the ratio of the observed and expected number of cases (O/E ratio).The external validations of PLCO-CA models were also measured as the AUCs and the O/E ratios of these models in the validation cohort.The optimal cut-off value of time-dependent ROC of 10-year PLCO cancer risk in the derivation cohort was used to classify participants into low-and high-risk groups.
Additionally, previous studies suggested that PLCO cancer screening can only significantly reduce the specific mortality of colorectal cancer (CRC) but not for other three cancers (9,(27)(28)(29).
To avoid the driving effect of CRC screening on joint cancer screening, further CRC-free sensitivity analyses were conducted to reassess the effects of joint cancer screening on all primary outcomes after excluding CRC cases (N=1656) and deaths (N=671) censored at the date of CRC incidence or mortality.
All statistical analyses were conducted via R software (version 4.1.0).The 'survival' package (version 3.3-1) was used to conducted survival analyses.The 'survminer' package (version 0.4.9) was used to draw survival curves.The 'survivalROC' package (version 1.0.3.1) was used to draw time-dependent ROC curve and calculate the area under ROC curve.The 'meta' package (version 5.5-0) was used to conduct meta analyses.The 'ggplot2' package (version 3.3.5)was used to draw the cancer stage graphs, and forest plots were drawn with 'forestplot' package (version 2.0.1).P value < 0.05 was considered statistically significant.

Result Baseline characteristics of participants between the screening arm and control arm
As shown in Table 1, in both males and females, compared with the control arm, the screening arm seemed to have younger age, fewer smokers, and fewer history of organ-related partial resection surgery.Some other differences can also be found between the gender-specific screening and control arms.For example, male participants in the screening arm had more family history of cancer compared to their counterparts in the control arm, while female participants in the screening arm used more hormones for menopause.However, none of the absolute differences in subgroup-specific proportions within each index variable between the screening arm and control arm was greater than 2%.Therefore, these differences could only be considered as statistical differences related with the specific selection flowchart rather than actually differences in baseline characteristics between the two arms.

Effectiveness of joint cancer screening on primary endpoints
After a median follow-up of 10.48 years for incidence and 16.85 years for mortality, a total of 5,506 PLCO cancer cases (4,636 males and 870 females), 1,845 PLCO cancer deaths (1,227 males and 618 females), 3,970 all-neoplasms deaths (2,576 males and 1,394 females), and 14,221 all-cause deaths (9,461 males and 4,760 females) were documented in the screening arm, while the numbers in the control arm were 6,261 (5,054 males and 1,207 females), 2,417 (1,531 males and 886 females), 5,091 (3,109 males and 1,982 females), and 18,516 (11,514 males and 7,002 females) in the control arm, respectively (Table 2).
Compared to the control arm, joint cancer screening significantly increased the percent of male early-stage (stage I and II) PLCO cancers across the whole study period (75.9%   1).Moreover, as observed in Figure 2, in both males and females, logrank tests showed a non-significant difference in PLCO cancer incidence between the screening arm and control arm (P values of 0.28 for males and 0.18 for females), but significantly lower PLCO cancer mortality (P values <0.001 for males and 0.029 for females), all-neoplasms mortality (P values <0.001 for males and 0.001 for females) and all-cause mortality (P values for both males and females <0.001) for the screening arm compared to the control arm.

Subgroup analyses on the effectiveness of risk-stratified joint cancer screening
As shown in Supplementary Figure 3, gender-specific PLCO-CA models showed good discrimination (AUCs of 0.60 (0.47, 0.64) for males and 0.71 (0.65, 0.73) for females) and good calibration(O/E ratios of 0.99(0.97,1.03) for males and 1.00 (0.93, 1.07) for females, both P values >0.05) in the derivation cohort.Similar discrimination (AUCs of 0.59(0.56,0.61) for males and 0.70 (0.65, 0.73) for females, both P values for differences between gender-specific derivation and validation AUCs >0.05) and calibration(both P values for O/E ratios >0.05 in males and females) were observed in the validation cohort.After reclassifying the participants into low-and high-risk subgroups according to the optimal cutoff value of ROCs for 10-year genderspecific PLCO-CA prediction risk.We observed similar genderspecific effectiveness of joint cancer screening in both low-and high-risk subgroups as the preliminary analyses (Table 2; Supplementary Figures 2, 4, 5).

FIGURE 3
Meta-analyses of gender-specific effects of joint cancer screening on primary endpoints.
Gender-specific effects of joint cancer screening on crude PLCO cancers incidence (A, E), PLCO cancers mortality (B, F), all neoplasms mortality (C, G), and all-cause mortality (D, H). (A, B, C, D) for males, and (E, F, G, H) for females.

Discussion
In this study based on PLCO cancer screening trial, joint cancer screening means that individuals in the screening group received multiple methods recommended by the guidelines to screen for different cancers during the same period.To our knowledge, based on the PLCO trial, this is the first study to investigate the effectiveness of joint cancer screening, and this is also the first intervention study to explore the potential benefits of risk-stratified joint cancer screening based on easy-to-use combined cancer risk assessment.According to previous results of the PLCO trial (9, 27-29), compared with the control group, four individual screenings on FIGURE 4 Sensitivity analyses on the effects of joint cancer screening by compliance.Group A was defined as complete compliance with the screening protocol.Group B was defined as never receiving any screening examinations or tests in the screening arm.PLCO cancer-specific incidence were non-significant 5% increase for lung cancer, significant 22% increase for prostate cancer, non-significant 21% increase for ovarian cancer and significant 21% decrease for colorectum cancer, while effects on cancer-specific mortality were non-significant 1% increase for lung cancer, non-significant 13% increase for prostate cancer, non-significant 18% increase for ovarian cancer and significant 26% increase for colorectum cancer.If there were no synergies and spillover effects between individual screenings, the effect sum of four individual screenings should be 27% increase for PLCO cancer incidence and 4% increase for PLCO cancer mortality, respectively.However, in this study, the pooled effects of joint screening compared to the control were non-significant 1% increase for cancer incidence and significant 11% decrease for cancer mortality, respectively.Both of them in this study were better than the simple effect sum of four individual screenings.For those with better adherence to joint screening, more benefits can be observed, with significant 38% and 36% decrease in cancer mortality for males and females, respectively.The joint decrease in cancer mortality would further climbed to 39% and 40% for high-risk males and females with better adherence.All of these might suggest a potential synergy between the individual screenings.Therefore, joint cancer screening should be recommended as a potentially more suitable choice to reduce the overall cancer burden than single cancer screening.Moreover, researches are needed in the future to explore and confirm the synergistic effect of joint cancer screening.In this study, PLCO joint cancer screening significantly reduce PLCO cancer mortality but did not reduce incidence.Two major reasons would lead to the non-significant reduction of PLCO cancer incidence after joint screening.First, only colorectal cancer had well-defined precancerous lesions, and other three PLCO cancers (prostate, lung, and ovarian cancer) had no precancerous lesions.Secondly, previous studies from the PLCO trial demonstrated that only FSG screening could effectively lead to early-stage shift of CRC (9), but no significant early-stage shift was observed for PSA combined with DRE screening for prostate cancer (28), CA125 combined with TVU screening for ovarian cancer (29), and chest radiograph screening for lung cancer (27).Additionally, nonadherence to screening protocol, contamination in the control arm, potential overdiagnosis associated with screening, and possible dilution of screening effect after long-term follow-up, might collectively result in non-significant reduction in PLCO cancer incidence (9,(27)(28)(29).Furthermore, joint screening significantly reduced male-specific PLCO cancer mortality, but not among females.Several reasons would lead to the genderspecific joint cancer screening effects.First, although men have a higher cancer incidence rate than women due to gender-specific hormonal, behavioral and genetic differences, female patients often present with higher-grade disease and experience worse outcomes (1,(33)(34)(35)(36)(37).Secondly, CRC screening has been observed to be less effective in women than in men (9,38), and lung cancer screening often demonstrates equal benefits between males and females (39).More studies are needed to validate the gender-specific joint cancer screening effects.
Joint cancer screening could not overcome the above limitations within a single cancer screening.On contrary, joint cancer screening may magnify these potential limitations into a relatively more obvious limitation.For example, the "all-versus-none" design of joint cancer screening would significantly increase the difficulty of implementing integrated interventions in the screening arm, which would likely to weaken the adherence to any single intervention.Participants in the control arm may also significantly increase the intentions of active seeking for any or all screening components within the joint cancer screening due to multiple informed consents, thereby increasing the likelihood of contamination in the control arm.Multiple false-positive results from joint cancer screening would likely to lead to more overexamination and over-diagnosis compared to a single false-positive result in the screening arm.Additionally, all participants in the joint cancer screening had to meet more indications for multiple screening exams compared to single cancer screening, hence increasing the bias of healthy volunteers.All these amplified limitations would collectively dilute the expected effectiveness of single cancer screening.
Even with the above-mentioned potentially amplified limitations, our analyses still showed a significant reduction in PLCO cancer mortality, all-neoplasms mortality, and all-cause mortality associated with joint cancer screening.Further CRCfree sensitivity analyses showed that joint cancer screening significantly increased the CRC-free PLCO cancer incidence and did not reduce the CRC-free PLCO cancer mortality, which were similar to the previous single cancer screening (27)(28)(29).However, more importantly, joint cancer screening still significantly reduced both CRC-free all-neoplasm mortality and all-cause mortality.These results suggested that there must be some important difference between joint and single cancer screening to offset and even overweigh limitations mentioned above.Although both joint cancer screening and four individual cancer screening use the similar methods recommended by the guidelines, combination of these methods scheduled at the same period would bring an additional health promotion effects compared with any single method scheduled in different periods due to the cumulative health concerns.When participants received any positive result in either joint or single cancer screening, their physicians would not only discuss with the participants to determine which diagnostic evaluation procedures would be performed, but also probably provide several health promotion suggestions on cancer prevention, such as quitting smoking, alcohol abstinence, exercising, weight control, adopting a healthy diet, increasing preventive medication (such as NSAIDs), and so on.Unlike a positive result in single cancer screening, multiple positive results from joint cancer screening would probably enhance more adoption of several health promotion behaviors due to the multiple suggestions by different physicians during the same period.The enhanced adoption of health promotion behaviors will not only prevent target cancers, but also prevent other cancers and chronic diseases (such as cardiovascular diseases) with shared risk factors (40)(41)(42)(43)(44). That's would be most likely reason why joint cancer screening could reduce the risks of all-neoplasm mortality and all-cause mortality.Additionally, multiple positive screening results would also strengthen the adherence to regular screening behavior due to the same cumulative health concerns.This may also partly explain why participants who completely followed multiple rounds of joint screening received more benefits in PLCO cancer mortality, all-neoplasms mortality, and all-cause mortality than those without good adherence to the multiple screening.
In addition to the health promotion difference between joint and single cancer screening modes, another two differences also deserved attentions.First, cost difference.This difference did not lie in the screening cost but in the logistic costs.Joint screening only requires one time of population recruitment, screening organization, information collection, etc., while single cancer screening requires independent organization for different cancers, thereby increasing the costs of screening organization.Second, effectiveness difference.For example, in a single lung cancer screening practice, residents who participate in screening may not end up dying from lung cancer, but may die from colorectal cancer, as the investigators primarily focus on lung cancer-related outcomes.The same risk would be observed in other single cancer screening.However, this risk would be significantly reduced in joint screening for lung and colorectal cancer.Although the frequency of screening varies for different cancers, most cancers are screened at intervals of 1, 2, 3 or 5 years.Therefore, the joint screening of different cancers can be well arranged in different intervals.In summary, due to the enhanced adoption of health promotion behaviors, potential reduced overall cost, and increased effectiveness, joint cancer screening scheduled at different cancer-specific intervals during the same period would be recommended to improve the overall cost-effectiveness of current single cancer screening and expected to reduce the overall cancer burden in the future.
Moreover, we developed gender-specific PLCO-CA models based on modifiable risk factors to predict the combined risk of prostate, lung, colorectal, and ovarian cancer.Although several guidelines have detailed cancer screening recommendation according to the age (13, 14, 45-47), this age-stratified screening recommendation could not effectively identify persons with other risk factors of cancer.Currently, several studies had suggested that risk-stratified screening strategy integrating multiple factors (both genetic and non-genetic factors) would be more effective in identifying people at high risk of cancer and improving the potential screening effect (48)(49)(50)(51).Moreover, consistent with previous studies (52), significant reduction of PLCO cancer mortality was only observed in participants at high risk of multiple cancer but not in those at low risk.All of these suggested that integrated risk-stratified screening scheme would be more important in joint cancer screening.However, as mentioned above, compared with single cancer screening, joint cancer screening not only requires more resources and manpower but also is relatively difficult to organize.Therefore, in resourcelimited settings, we still recommend joint cancer screening for participants at high risk of multiple cancers or pan-cancer.In addition, although the AUCs of the gender-specific PLCO-CA models in this study were relatively good, there is still much room to improve the overall prediction discrimination.Further studies have confirmed that integrating polygenic risk scores with modifiable risk factors improves risk prediction of pan-cancer (53).Moreover, liquid biopsy markers, such as methylation signatures in cell-free DNA, are expected to have a huge potential value in early detection of pan-cancer (54)(55)(56).
Last but not the least, as in the PLCO trial, the joint cancer screening has been initiated in some developed countries.For example, a 55-year-old female smoker in the United States will be regularly screened for breast cancer (mammogram), cervical cancer (HPV test and/or a pap test), colorectal cancer (colonoscopy or stool-based test), and lung cancer (low-dose CT scan), which are initiated by her primary care physician.However, primary care in most other countries does not cover all these screening tests for above cancers.Therefore, integrating the scattered single cancer screening resources into a unified joint screening model would be a relatively optimal choice advocated in countries where joint screening is not yet available.Second, in addition to the cancers jointly screened in the United States, it is important to designate a country-specific joint cancer screening program based on their cancer burden priority.Third, joint cancer screening is not just the combination of conventional tests screening for different cancers separately, it could also be a unified method, such as liquid biopsy, to screen for different cancers.Fourth, since the synergistic effect of joint screening would probably attributable to non-cancer specific effects, the effects of cancer-specific screening between joint screening and single screening are probably not the same.Future RCTs with parallel arms are needed to investigate this difference and further support the synergistic effect of joint screening.
In addition to the several strengths above, there are also several limitations in this study.First, as the PLCO trial was the only published RCT for joint cancer screening in the world until now, there is currently no suitable study for external validation.Although both subgroup analyses and sensitivity analyses yielded very similar effects of joint cancer screening, the current results are based on one RCT study with specific population groups and specific age group.Therefore, it could be expected the current findings would not be broadly applicable to other population groups.Second, changes in health behavior after multiple positive screenings may be an important factor associated with all-neoplasms mortality and allcause mortality, especially adoption of healthier lifestyle after positive cancer screens.Future studies with more sophisticated designs and more exploration are needed to further support this hypothesis.Third, joint cancer screening is expected to be more cost-effective than single cancer screening.However, the PLCO trial did not collect the data needed for health economic evaluation.Additionally, the long latency period, high disease prevalence, and significant associated morbidity and mortality are the three basic criteria for risk-reduction cancer screening and intervention.Latency period significantly varies across different cancers.According to a recent study, the estimated median latency period of lung, breast, ovarian, and rectum cancers was 13.6, 16.3, 44.1, and 29.8 years, respectively (57).Moreover, the latency period of cancer would be modulated by different risk factors, such as smoking, ionizing radiation exposure, special occupation exposure, and use of cancer-prevention medicine (58)(59)(60).Although a median follow-up of 10.48 years for incidence and 16.85 years for mortality were recorded in this study, it would not be long enough to capture meaningful outcomes.

Conclusion
Based on the unique "all-versus-none" RCT design, joint cancer screening can significantly reduce PLCO cancer mortality, allneoplasms mortality, and all-cause mortality.More compliance, more benefits.Therefore, joint cancer screening could be recommended as a potentially more suitable choice to reduce the overall cancer burden than traditional single cancer screening.However, notably, organizing a joint cancer screening not only requires more resources, manpower and ingenious design, but also needs more attentions to the potential harms.Although the current results support that joint cancer screening can also benefits the lowrisk populations, we still prioritize joint cancer screening for highrisk groups to reduce the difficulty of joint intervention and improve the potential cost-effectiveness.The author(s) declare financial support was received for the research, authorship, and/or publication of this article.This work was supported by the Chinese National Key Research and Development Project (No. 2021YFC2500400), Tianjin Health Committee Foundation (No. TJWJ2021MS008) and National Natural Science Foundation of China (81974439).The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

FIGURE 1
FIGURE 1Gender-specific effects of joint cancer screening on cancer stage shift.Missing data of cancer stage were not included in this figure.

TABLE 1
Comparison of baseline characteristics between the screening arm and the control arm by gender.
BMI, body mass index.HRT, hormone replace therapy.

TABLE 2
Gender-specific effects of joint cancer screening on primary endpoints by risk groups.PYs, person years.*, adjusting age, smoking, diabetes, prostate surgery, family history.¶, adjusting age, smoking, diabetes, ovary surgery, organ-related disease, oral contraception and family history.The bold values in Table 2 indicate P values of less than 0.05.